We have convincingly shown that natural killer (NK) cells facilitate the pathogenesis of sepsis caused by cecal ligation and puncture (CLP). However, the mechanisms that contribute to NK cell-mediated pro-inflammatory activity during sepsis are poorly understood. Our recent studies indicate large numbers of CXCR3+ NK cells leave the spleen and enter the peritoneal cavity during CLP-induced sepsis. The CXCR3 ligands CXCL9 and CXCL10 are present at high concentrations during sepsis and CXCR3-deficient mice are resistant to CLP-induced physiologic dysfunction. Based on these observations, we hypothesize that CXCR3 signaling is critical for the recruitment and activation of NK cells and that the actions of CXCR3 are important in the pathogenesis of sepsis. The following specific aims will test these hypotheses. Specific Aim 1: To determine the importance of CXCR3 for NK cell migration and activation as well as its impact on the pathogenesis of CLP- induced sepsis. The expression of CXCR3 and markers of activation will be evaluated on NK cells at the primary site of infection and in remote tissues. Further studies will assess NK cell recruitment and activation in CXCR3-deficient mice and after blockade of CXCR3 with neutralizing antibodies. We will also determine the importance of CXCR3 in the pathogenesis of sublethal sepsis, sepsis-induced multi- organ dysfunction and septic shock. Specific Aim 2. To determine the importance of CXCR3 ligands (CXCL9 and CXCL10) for NK cell recruitment and activation as well as their impact on the pathogenesis of CLP-induced sepsis. The cellular sources of CXCR3 ligands at the primary site of infection and remote tissues will be examined. NK cell migration and activation will be studied in CXCR3 ligand-deficient mice or after blockade of CXCR3 ligands using neutralizing antibodies. Physiological function, organ injury and systemic inflammation will be examined in CXCR3 ligand- deficient mice using models of sublethal sepsis, sepsis-induced multi-organ dysfunction and septic shock. We will also determine the ability of CXCR3 ligands to directly induce the sepsis syndrome in control mice or mice with sublethal sepsis. Specific aim 3. Evaluation of factors that regulate the CXCR3 axis during CLP-induced sepsis. These studies will examine the contributions of NK cells to CXCR3 ligand production and the factors that regulate NK cell CXCR3 expression during sepsis. Although LPS-induced CXCL10 production by isolated macrophages is considered to be regulated by Trif-dependent signaling and require production of IFN2, we propose that NK cells will facilitate MyD88- dependent CXCL10 production through the production of IFN3. Studies are proposed in this application to address that assertion. Further studies will evaluate the importance of paracrine and autocrine mechanisms for NK cell CXCR3 activation and the factors that regulate CXCR3 expression by NK cells. PUBLIC HEALTH RELEVANCE: We have convincingly shown that natural killer (NK) cells facilitate the pathogenesis of sepsis. The chemokine receptor CXCR3 is known to regulate NK cell functions in some disease models. Based on our recent research, we hypothesize that CXCR3 signaling is critical for the recruitment and activation of NK cells during sepsis and that blockade of CXCR3 will improve outcome in septic animals.